Antistasin, a 15 kDa cysteine rich (20 cysteines of 119 amino acids) protein found in the salivary gland of the Mexican leech, Haementeria officinalis, has been shown to be a potent inhibitor of Factor Xa in the human blood coagulation cascade, Tuszynski et al., J. Biol. Chem. 262: 9718-9723 (1987); Nutt et al., J. Biol. Chem. 263: 10162-10167 (1988). Antistasin's ability to inhibit Factor Xa's activity, which can be activated by both the intrinsic and extrinsic pathways, makes it an attractive candidate for anticoagulant therapy. Furthermore, because antistasin inhibits the coagulation cascade at a step earlier than those anticoagulants acting on thrombin, such as hirudin and heparin, it has the potential of being a more efficient anticoagulant.
Sequence analyses show that antistasin possesses an internal homology between roughly the N-terminal and the C-terminal halves (40% for amino acids and 50% for nucleotides), Nutt et al., J. Biol. Chem. 263: 10162-10167 (1988); Han et al., Gene (Amst.) 75: 47-57 (1989). Both the N- and C-terminal halves contain 10 cysteines and each cysteine residue's location is conserved in each half.
It has been demonstrated previously that antistasin is cleaved following Factor Xa binding. This cleavage, which occurs in the N-terminal half at arg 34, is presumed to be caused by the binding of Factor Xa (Dunwiddie et al., J. Biol. Chem. 264: 16694-16699 (1989)).
This invention includes the construction and production of a protein having a high degree of homology with antistasin N-terminal amino acids 1 through 58. This protein is biologically active and retains a significant portion of the Factor Xa inhibitory activity of the whole antistasin molecule.